Method for preparing nicotianamine or nicotianamine-containing product

ABSTRACT

Precipitation fractionation of an aqueous extract of soybeans by addition of an organic solvent such as ethanol or molecular weight fractionation of the extract by ultrafiltration or size exclusion chromatography is appropriately combined with activated carbon filtration, cation- or anion-exchange resin treatment or other adsorbent (e.g., polyamide or octadecylsilica) treatment to provide a nicotianamine product of desired purity. The product is added to food and drink to provide health foods.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a method for preparingnicotianamine or a nicotianamine-containing product which hasangiotensin converting enzyme (ACE) inhibitory activity from an aqueousextract of soybeans and to a health food containing thenicotianamine-containing product as a functional ingredient.

[0003] 2. Description of the Related Art

[0004] Nicotianamine has blood pressure lowering action based on ACEinhibition. Nicotianamine is widely found in the vegetable world, suchas tobaccos, rice plants, Chinese matrimony vines, and beeches, and areobtainable from the leaves of these plants (JP-A-63-87990). It is knownthat nicotianamine is also found in kidney beans and soybeans and thatan extract of the beans with water or hot water is treated with asynthetic resin to obtain purified nicotianamine (JP-A-5-246865).

[0005] However, the method of obtaining nicotianamine through extractionof plant leaves meets difficulty in stably securing a large quantity ofthe plant. Since production of kidney beans is very low, it tends to bedifficult to procure a stable supply as a raw material of nicotianamine.

[0006] On the other hand, soybeans are stably supplied. However, wherenicotianamine is prepared from an aqueous extract of soybeans, such as aboiling waste liquid or a soaking waste liquid in the soybean processingindustry, by adsorption and desorption by a synthetic resin, the yieldis low, resulting in a high cost.

SUMMARY OF THE INVENTION

[0007] The inventors studied seeking a method of efficiently preparingnicotianamine from soybeans or soybean materials. As a result they havefound that addition of an organic solvent to an aqueous extract ofsoybeans results in formation of a precipitate, which containsnicotianamine in a high concentration and that a fraction collected froman aqueous extract of soybeans which has a molecular weight of 1,000 orless contains nicotianamine in a high concentration. They have alsofound that nicotianamine can be obtained with an economic advantage byutilizing, as an aqueous extract, soybean whey which is by-produced inthe production of soybean protein isolate.

[0008] Based on these findings, the present invention provides a methodof preparing nicotianamine comprising adding an organic solvent to anaqueous extract of soybeans and purifying the resulting precipitate anda method of preparing a nicotianamine-containing product comprisingadding an organic solvent to an aqueous extract of soybeans andcollecting the resulting precipitate as a crude product. The presentinvention also provides a method of preparing nicotianamine or anicotianamine-containing product comprising subjecting an aqueousextract of soybeans to ultrafiltration or size exclusion chromatographyto collect a fraction having a molecular weight of 1,000 or less. Thepresent invention further provides a method for preparing anicotianamine-containing product having a nicotianamine content of 0.3%by weight or more which comprises subjecting an aqueous extract ofsoybeans to ultrafiltration or size exclusion chromatography, collectinga fraction having a molecular weight of 1,000 or less, adding an organicsolvent to the fraction, and collecting the resulting precipitate. Thepresent invention furthermore provides a health food containing thenicotianamine-containing product as a functional ingredient.

[0009] According to the methods of the present invention, nicotianamineor a nicotianamine-containing product having ACE inhibitory activity isprepared efficiently from an aqueous extract of soybeans, particularlysoybean whey. The resulting nicotianamine or nicotianamine-containingproduct can be taken as such as a health food or be added to foods toprovide health foods for treating or preventing symptoms ofhypertension.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Soybeans which can be used in the present invention include wholesoybeans, skinned soybeans, ground soybeans, and defatted soybeans usedas a raw material of soy sauce. The aqueous soybean extract includes anextract of soybeans with water or hot water, a waste liquid used forsoaking soybeans, a waste liquid used for boiling soybeans in thesoybean processing industry, soybean whey by-produced in the manufactureof soybean protein isolate, and soybean whey by-produced in themanufacture of tofu (soybean curd).

[0011] The aqueous soybean extract may be used as such, but it isadvisable to previously remove dissolved proteins because the proteinsmay cause reduction in purity or ACE inhibitory activity ofnicotianamine. The proteins can be removed in a conventional manner, forexample, by adjusting the pH of the extract to about 4.5 to cause themto precipitate.

[0012] A suitable aqueous extract of soybeans is prepared by soakingsoybeans in 1 to 20 times their weight of water or hot water for atleast 5 minutes, preferably 1 to 20 hours. The extraction is preferablyconducted at pH 7 to 9 for extraction efficiency.

[0013] An acid is added to the resulting aqueous extract to adjust thepH value to about 4.5, and the extract is centrifuged to remove theprecipitate. The supernatant is a preferred material. It is particularlyadvantageous from the standpoint of utilization of resources to usesoybean whey which is by-produced in the production of soybean proteinisolate and has mostly been discarded.

[0014] If desired, the aqueous soybean extract is concentrated, and anorganic solvent is added thereto. A water-soluble organic solvent, suchas methanol, ethanol, and acetone, is used. Ethanol is particularlypreferred. The organic solvent is preferably added in a concentration of30% (by weight, hereinafter the same) or more, particularly 50% or more,while the amount varies depending on the degree of concentration of theextract.

[0015] Upon addition of the organic solvent, a precipitate is formed inthe aqueous extract, which is then collected by an appropriate means,such as centrifugation. The soybean aqueous extract concentrated to ahigher degree tends to form a precipitate easier at a lowerconcentration of the organic solvent. For example, where soybean wheyobtained by extraction with 10 times the weight of water is used assuch, a suitable organic solvent concentration is 70 to 90%, while a 3-to 4-fold concentrate of the whey produces a sufficient precipitate atan organic solvent concentration of 50 to 60%.

[0016] The precipitate as collected, which contains nicotianamine in ahigh concentration, is dried to powder by vacuum drying, lyophilization,or spray drying to obtain a nicotianamine crude product, which canfurther be purified to provide a nicotianamine purified product.

[0017] It might be conceivable to directly dry an aqueous extract ofsoybeans, such as whey, to powder, but this involves disadvantagesarising from various substances dissolved in the extract such that theresulting nicotianamine product has nicotianamine of low purity, and theextract is difficult to dry to powder or fails to provide satisfactorypowder.

[0018] The crude nicotianamine product can be purified by, for example,the method described in JP-A-5-246865, Example 2. The physicochemicalproperties of the nicotianamine thus prepared are as described inJP-A-5-246865, paragraph 0014.

[0019] Impurities can further be reduced by treating the aqueous soybeanextract by ion-exchange resin treatment, activated carbon filtration oradsorbent treatment or a combination thereof either before or after theorganic solvent addition.

[0020] Being ampholytic, nicotianamine can be treated with either acation-exchange resin or an anion-exchange resin. Since nicotianaminehas high polarity and is thereby substantially inert to activatedcarbon, treatment with activated carbon removes oligosaccharides or likeimpurities present in soybean whey. To remove organic matter ofrelatively low polarity, such as isoflavons and saponins, the extractcan be treated with octadecylsilica, polyamide, synthetic adsorbents(e.g., Diaion HP-20, available from Mitsubishi Chemical Corp.), etc.

[0021] Nicotianamine, the molecular weight of which is 303, is able topass through an ultrafiltration membrane having a molecular weightcut-off of about 1,000 even when it is in the form of a salt. Whensubjected to size exclusion chromatography, it can be collected in afraction of molecular weight 1,000 or less. Therefore, it is alsopossible to recover nicotianamine or a nicotianamine-containing productfrom an aqueous soybean extract such as soybean whey by ultrafiltrationor size exclusion chromatography to collect a fraction having amolecular weight of 1,000 or less.

[0022] Nicotianamine or a nicotianamine-containing product with higherpurity can be obtained by treating the aqueous soybean extract byion-exchange resin treatment, activated carbon filtration or adsorbenttreatment or a combination thereof either before or afterultrafiltration or size exclusion chromatography as described above.

[0023] The nicotianamine-containing product of the present invention canbe desalted by electrodialysis or a like means. The dialyzate, whichcontains nicotianamine in a high concentration, is dried to powder byvacuum drying, lyophilization or spray drying to obtain a crudenicotianamine product, which can further be purified by theabove-mentioned purification techniques to prepare a purifiednicotianamine product. Purification can be effected to a desired degreeaccording to the intended use of the product.

[0024] The above-described method comprising adding an organic solventand the method comprising ultrafiltration or size exclusionchromatography can be combined to provide a nicotianamine-containingproduct having a nicotianamine content of 0.3% or more. In this case,also, the above-described treatment with an ion-exchange resin,activated carbon or an adsorbent or a combination thereof can beperformed either before or after addition of an organic solvent oreither before or after ultrafiltration or size exclusion chromatography.For example, an aqueous soybean extract, preferably soybean whey, istreated by activated carbon filtration, and the filtrate is subjected toultrafiltration or size exclusion chromatography to harvest a fractionhaving a molecular weight of 1,000 or smaller. An organic solvent isthen added to the fraction in a concentration of 30%, and theprecipitate thus formed is collected to give a nicotianamine-containingproduct having a nicotianamine content of 0.3% or more.

[0025] The present invention also provides a health food containing thenicotianamine or nicotianamine-containing product (a crude product or apurified product) obtained by the method of the invention as afunctional ingredient. For example, the crude nicotianamine-containingproduct is mixed with common vehicles and punched into tablets orincorporated into a variety of foods and beverages, such as bakeryproducts (e.g., bread, cake, and cookies), noodles (e.g., buckwheatnoodle, wheat noodle, and Chinese noodle), cooked foods (e.g., tempuraor deep-fried foods), soybean foods (e.g., tofu and natto (fermentedsoybeans)), juice, coffee, cocoa, woolong tea, green tea, and healthdrinks, to make functional food and drink having antihypertensiveactivity of nicotianamine.

[0026] The present invention will now be illustrated in greater detailwith reference to Examples. Unless otherwise noted, all the percents andparts are by weight.

EXAMPLE 1

[0027] To 400 g of defatted soybean meal was added 5 l of water, andwater was adjusted to pH 9. After 90-minute soaking at 25° C., thesoybean meal was separated to obtain 4.7 l of the aqueous extract. Theextract was adjusted to pH 4.5 and centrifuged at 7500 G for 30 minutesto obtain 4.5 l of the supernatant liquid (soybean whey). Ethanol wasadded to the whey to a concentration of 80%, followed by centrifugationat 4° C. and at 7500 G for 30 minutes. The collected precipitate waslyophilized to give 12 g of a crude nicotianamine product as whitepowder, which was found to have a nicotianamine content of 0.5%.

EXAMPLE 2

[0028] To 4.5 l of soybean whey prepared in the same manner as inExample 1 was added 150 g of activated carbon (Shirasagi, available fromTakeda Chemical Industries, Ltd.), and the mixture was stirred at roomtemperature for 3 hours to have impurities adsorbed onto the activatedcarbon. Into the mixture was mixed 150 g of a diatomite filter aid(Radiolite 500), and the mixture was filtered by suction through filterpaper No. 2. The filtrate was treated through a hollow fiber typeultrafiltration membrane module having a molecular weight cut-off of1,000 (Prepscale UF Cartridge, PLAC type, available from Nihon MilliporeK.K.). The ultrafiltrate was lyophilized to give 30 g of a white powder,which was found to have a nicotianamine content of 0.3%.

EXAMPLE3

[0029] The powder obtained in Example 2 was re-dissolved in 200 ml ofdistilled water, and ethanol was added to a concentration of 80%. Theethanol solution was centrifuged at 4° C. and at 7500 G for 30 minutes.The collected solid was lyophilized to obtain 5.4 g of a crudenicotianamine product as a white powder, which contained 1.0% ofnicotianamine.

EXAMPLE 4

[0030] The ultrafiltrate obtained in Example 2 was passed through a 1-lcolumn of an anion-exchange resin (Diaion PA318, OH⁻ form, availablefrom Mitsubishi Chemical Corp.). The column was washed with 2 l ofdistilled water and eluted with 2 l of 0.5 N hydrochloric acid. Theeluate was further passed through a 1-l column of a cation-exchangeresin (Dowex 50W×2, H⁺ form, available from the Dow Chemical Co.), andthe column was washed with 2 l of distilled water and eluted with 0.5%aqueous ammonia. The fraction of from 2 l up to 3 l was collected andconcentrated to 10 ml on a rotary evaporator. The concentrate waslyophilized to give 100 mg of a white powder, which had a nicotianaminecontent of 40%.

EXAMPLE 5

[0031] The nicotianamine-containing powder obtained in Example 4 wasdissolved in a small amount of water, and the solution was passedthrough a 0.5-l column of polyamide C-200 (available from Wako PureChemical Industries, Ltd.) pre-equilibrated with 100% ethanol and elutedsuccessively with a 1 liter portion each of 100% ethanol, 90% ethanol,80% ethanol, 70% ethanol, 60% ethanol and 50% ethanol. The eluate with70% ethanol, 60% ethanol and 50% ethanol was concentrated, filtered, andlyophilized to give 40 mg of a white powder, which was found to have anicotianamine content of 80%.

EXAMPLE 6

[0032] Two liters of water was added to 400 g of a waste soybean boilingliquid and the obtained mixture was kept at 25° C. for 90 minuets. Theinsoluble materials were removed by centrifugation to leave 1.9 l of thesupernatant liquid. Ethanol was added to the liquid in a concentrationof 80%, and the mixture was centrifuged at 4° C. and at 7500 G for 30minutes. The separated precipitate was lyophilized to give 4 g of acrude nicotianamine product as a white powder, which was found to have anicotianamine content of 0.15%.

EXAMPLE 7

[0033] The following components were mixed up uniformly into granulesand punched in a conventional manner to prepare tablets each weighing500 mg. Crude nicotianamine powder prepared in Example 1 300 g Lactose100 g Crystalline cellulose  93 g Talc  5 g Carboxymethyl cellulose  2 g

EXAMPLE 8

[0034] The following components were compounded and baked to make cakein a usual manner. Wheat flour 100 parts Egg 100 parts Sugar 100 partsCrude nicotianamine powder prepared in Example 1  3 parts Water  40parts

[0035] The cake was satisfactory in palatability and flavor with nodifference from one containing no nicotianamine product in both textureand appearance.

EXAMPLE 9

[0036] The crude nicotianamine powder prepared in Example 1 was added toa commercially available carbonized beverage in a concentration of 1%.It dissolved quickly, and the resulting beverage presented no change inflavor.

[0037] The invention being thus described, it will be obvious that thesame may be varied in many ways. Such variations are not to be regardedas a departure from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

[0038] This application claims the priority of Japanese PatentApplication No. 2000-375403 filed Dec. 11, 2000, which is incorporatedherein by reference.

What is claimed is:
 1. A method of preparing nicotianamine comprisingadding an organic solvent to an aqueous extract of soybeans andpurifying the resulting precipitate.
 2. The method of preparingnicotianamine according to claim 1, wherein said aqueous extract ofsoybeans is soybean whey.
 3. The method of preparing nicotianamineaccording to claim 1, wherein said organic solvent is added to aconcentration of 30% by weight or more.
 4. The method of preparingnicotianamine according to claim 1, wherein said organic solvent hassolubility in water.
 5. The method of preparing nicotianamine accordingto claim 1, which further comprises subjecting the aqueous extract to atleast one of ion-exchange resin treatment, activated carbon filtrationand other adsorbent treatment either before or after the addition of theorganic solvent.
 6. A method of preparing a nicotianamine-containingproduct comprising adding an organic solvent to an aqueous extract ofsoybeans and collecting the resulting precipitate as a crude product. 7.The method of preparing a nicotianamine-containing product according toclaim 6, wherein said aqueous extract of soybeans is soybean whey. 8.The method of preparing a nicotianamine-containing product according toclaim 6, wherein said organic solvent is added to a concentration of 30%by weight or more.
 9. The method of preparing a nicotianamine-containingproduct according to claim 6, wherein said organic solvent hassolubility in water.
 10. The method of preparing anicotianamine-containing product according to claim 6, which furthercomprises subjecting the aqueous extract to at least one of ion-exchangeresin treatment, activated carbon filtration and other adsorbenttreatment either before or after the addition of the organic solvent.11. A health food containing, as a functional ingredient, a precipitateformed by adding an organic solvent to soybean whey to a concentrationof 30% by weight or more.
 12. A method of preparing nicotianamine or anicotianamine-containing product comprising subjecting an aqueousextract of soybeans to ultrafiltration or size exclusion chromatographyto collect a fraction having a molecular weight of 1,000 or less. 13.The method of preparing nicotianamine or a nicotianamine-containingproduct according to claim 12, wherein said aqueous extract of soybeansis soybean whey.
 14. The method of preparing nicotianamine or anicotianamine-containing product according to claim 12, which furthercomprises subjecting the aqueous extract to at least one of ion-exchangeresin treatment, activated carbon filtration and other adsorbenttreatment either before or after the ultrafiltration or size exclusionchromatography.
 15. A health food containing, as a functionalingredient, a fraction having a molecular weight of 1,000 or less whichis collected by subjecting soybean whey to ultrafiltration or sizeexclusion chromatography.
 16. A method of preparing anicotianamine-containing product having a nicotianamine content of 0.3%by weight or more which comprises subjecting an aqueous extract ofsoybeans to ultrafiltration or size exclusion chromatography to collecta fraction having a molecular weight of 1,000 or less, adding an organicsolvent to the fraction, and collecting the resulting precipitate. 17.The method of preparing a nicotianamine-containing product according toclaim 16, which further comprises subjecting the aqueous extract or thefraction to at least one of ion-exchange resin treatment, activatedcarbon filtration and other adsorbent treatment either before or afterthe ultrafiltration or size exclusion chromatography or the addition ofthe organic solvent.
 18. A method of preparing anicotianamine-containing product having a nicotianamine content of 0.3%by weight or more which comprises subjecting an aqueous extract ofsoybeans to activated carbon filtration, subjecting the filtrate toultrafiltration or size exclusion chromatography to collect a fractionhaving a molecular weight of 1,000 or less, adding an organic solvent tothe fraction in a concentration of 30% by weight or more, and collectingthe resulting precipitate.
 19. The health food containing thenicotianamine-containing product prepared by the method of claim 16 or18 as a functional ingredient.